RNF12 initiates X-chromosome inactivation by targeting REX1 for degradation

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Abstract

Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In placental mammals, this is achieved by random inactivation of one X chromosome in all female somatic cells1. Upregulation of Xist transcription on the future inactive X chromosome acts against Tsix antisense transcription, and spreading of Xist RNA in cis triggers epigenetic changes leading to X-chromosome inactivation. Previously, we have shown that the X-encoded E3 ubiquitin ligase RNF12 is upregulated in differentiating mouse embryonic stem cells and activates Xist transcription and X-chromosome inactivation2. Here we identify the pluripotency factor REX1 as a key target of RNF12 in the mechanism of X-chromosome inactivation. RNF12 causes ubiquitination and proteasomal degradation of REX1, and Rnf12 knockout embryonic stem cells show an increased level of REX1. Using chromatin immunoprecipitation sequencing, REX1 binding sites were detected in Xist and Tsix regulatory regions. Overexpression of REX1 in female embryonic stem cells was found to inhibit Xist transcription and X-chromosome inactivation, whereas male Rex1+/− embryonic stem cells showed ectopic X-chromosome inactivation. From this, we propose that RNF12 causes REX1 breakdown through dose-dependent catalysis, thereby representing an important pathway to initiate X-chromosome inactivation. Rex1 and Xist are present only in placental mammals, which points to co-evolution of these two genes and X-chromosome inactivation.

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Figure 1: RNF12 interacts with REX1 in mouse ESCs.
Figure 2: RNF12 polyubiquitinates and targets REX1 for proteasomal degradation.
Figure 3: RNF12 is a dose-dependent regulator of REX1 expression.
Figure 4: REX1-dependent regulation of XCI.

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Acknowledgements

We thank R. van der Linden and C. Maduro for their help with some of the experiments, A. Inagaki, C. Andrieu-Soler, D. Warmerdam, L. Zhang and A. Mohd-Sarip for experimental advice, and C. Rougeulle and R. Jaenisch for providing the Tsix-stop cells. This work was supported by grants from the Netherlands Organisation for Scientific Research (NWO-TOP and NWO-VICI) and the European Research Council to J.G.

Author information

C.G. and J.G. designed the experiments. C.G. performed most experiments assisted by E.R. T.S.B., C.G. and J.G. generated the Rnf12−/− ES cells line. C.G., J.D., W.v.IJ. and E.M.A. performed the mass spectrometry and sequencing analysis. J.G., J.A.G. and C.G. wrote the manuscript.

Correspondence to Joost Gribnau.

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